ABSTRACT Both egg- and cell-adapted canine distemper virus (CDV) vaccines are suspected to retain residual virulence, especially if administered to immuno-suppressed animals, very young pups or to highly susceptible animal species. In the early 1980s, post-vaccine encephalitis was reported in dogs from various parts of Britain after administration of a particular batch of combined CDV Rockborn strain/canine adenovirus type-1 vaccine, although incrimination of the Rockborn strain was subsequently retracted. Notwithstanding, this, and other reports, led to the view that the Rockborn strain is less attenuated and less safe than other CDV vaccines, and the Rockborn strain was officially withdrawn from the markets in the mid 1990s. By sequencing the H gene of the strain Rockborn from the 46th laboratory passage, and a commercial vaccine (Candur(®) SH+P, Hoechst Rousell Vet GmbH), the virus was found to differ from the commonly used vaccine strain, Onderstepoort (93.0% nt and 91.7% aa), and to resemble more closely (99.6% nt and 99.3% aa) a CDV strain detected in China from a Lesser Panda (Ailurus fulgens). An additional four CDV strains matching (>99% nt identity) the Rockborn virus were identified in the sequence databases. Also, Rockborn-like strains were identified in two vaccines currently in the market. These findings indicate that Rockborn-like viruses may be recovered from dogs or other carnivores with distemper, suggesting cases of residual virulence of vaccines, or circulation of vaccine-derived Rockborn-like viruses in the field.

[Show abstract][Hide abstract]ABSTRACT:
Canine distemper virus (CDV) is a highly contagious viral disease of carnivores affecting both wild and domestic populations. The hemagglutinin gene, encoding for the attachment protein that determines viral tropism, shows high heterogeneity among strains, allowing for the distinction of ten different lineages distributed worldwide according to a geographic pattern. We obtained the sequences of the full-length H gene of 15 wild-type CDV strains circulating in domestic dog populations from the Aburrá Valley, Colombia. A phylogenetic analysis of H gene nucleotide sequences from Colombian CDV viruses along with field isolates from different geographic regions and vaccine strains was performed. Colombian wild-type viruses formed a distinct monophyletic cluster clearly separated from the previously identified wild-type and vaccine lineages, suggesting that a novel genetic variant, quite different from vaccines and other lineages, is circulating among dog populations in the Aburrá Valley. We propose naming this new lineage as "South America 3". This information indicates that there are at least three different CDV lineages circulating in domestic and wild carnivore populations in South America. The first one, renamed Europe/South America 1, circulates in Brazil and Uruguay; the second, South America 2, appears to be restricted to Argentina; and the third, South America 3, which comprises all the strains characterized in this study, may also be circulating in other northern countries of South America.

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We characterized two recent Japanese isolate CDV strains.•The Yanaka strain was avirulent, while the Bunkyo-K strain was virulent in dogs.•We tested the ability of the Yanaka strain for vaccination.•The Yanaka strain efficiently induced protective immunity against virulent CDVs.•The Yanaka strain is a potential candidate for a novel live attenuated CDV vaccine.

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V. Martella et al. / Vaccine 29 (2011) 1222–12271223but became ‘completely attenuated’ after 56 serial passages [44];it was found to loose immunogenicity by the 70th passage. Thevaccine was distributed globally after 1962 and proved to elicitsolid immunity and protection to vaccinated animals. In the early1980s, post-vaccinal encephalitis was reported in dogs from var-ious parts of Britain after administration of a particular batch ofcombined CDV Rockborn strain/canine adenovirus type-1 vaccine,although incrimination of the Rockborn strain was subsequentlyretracted [13,14]. This report noted analogous observations madein 1974 and 1978 [4,27] and led to the opinion that the RockbornstrainwaslessattenuatedandlesssafethanotherCDVvaccines,e.g.Onderstepoort strain. Suspected cases of post vaccinal encephali-tis were also reported in 1995 in USA in dogs vaccinated with apolyvalent vaccine containing the strain Rockborn. Consequently,the Rockborn strain was withdrawn from several markets after themid 1990s [18].The H glycoprotein is a key protein for CDV as the virus usesthis protein for attachment to the signaling lymphocyte activationmolecule (SLAM) receptor (CD150) [49] on the cell membrane andan adequate host immune response against the H protein may pre-vent CDV infection [53]. Comparative studies of CDV strains haverevealed that the H gene is subjected to higher genetic/antigenicvariation than other CDV genes and it appears as the most suit-able target to obtain epidemiological information [7,22,31,36,40].In spite of its historical importance, only limited sequence infor-mation is available for the Rockborn strain, and the sequence of theH gene has not been determined. Sequence information on the Hgene is available for the avianized strains Onderstepoort and Led-erle [11]. Sequence information is also available for the pathogenicSnyder Hill virus [9], but not for the ‘attenuated Snyder Hill virus’,that seems to be no longer available in the market. Based on theH gene, the vaccine strains Ondestepoort, Lederle and Snyder Hillappear to be genetically related to each other (<4% aa variation)but more distantly related (>8% aa variation) to the CDV strainsspreadingworldwide[8,12,22,26,30,34,37,40].Takingadvantageofthisgenetic/antigenicdiversity,severaldiagnosticassayshavebeendeveloped that are able to distinguish between the Onderstepoortstrain and field CDV strains [31,40–42,50,36].In order to fill this information gap, the sequence of the H geneof a laboratory passage (46th) of the vaccine CDV strain Rockborn,available from the James Baker Institute, was determined. In addi-tion, the sequence of commercially available CDV vaccines wasdetermined and compared.1. Materials and methods1.1. Virus originThe 46th laboratory passage of the vaccine CDV strain Rockborn(Rockborn-46th) was available at the James Baker Institute labora-tories. The virus was re-amplified by two serial passages on VEROcells and viral growth was monitored by visualization of the cyto-pathiceffectandbyanindirectimmunofluorescence(IF)assay.Thevaccine Candur®SH+P (Hoechst Rousell Vet GmbH) (batch number412684)thatcontainsthestrainRockborn(Rockborn-Candur),wasalso analysed.Two additional vaccines (A and B) commercialized from twodifferent companies were also analysed. For those vaccines, infor-mation on the CDV strain was not specified on the labels. Byusing an RT-PCR genotyping assay with primers specific for vari-ous CDV genotypes (America-1, Arctic-like, European, Asia-1 andAsia-2) [36] vaccines A and B were found not to contain America-1(Onderstepoort-like) CDV strains. Therefore, the sequence of the Hgene was determined in order to establish firmly which CDV strainwas included in the vaccine formulation.1.2. RNA extraction, reverse transcription and PCR amplificationA 140?l lysate of cells infected with strain Rockborn-46th and 140?l of each vaccine suspension were used forRNA extraction. The RNA was extracted using the RNeasy Kit(Qiagen,GmbH,Germany)accordingtothemanufacturer’sinstruc-tions.ReversetranscriptionandPCRamplificationoftheHgeneofCDVwas achieved as previously described [37]. To obtain a PCR prod-uct for sequencing of the H gene of the Italian strains, 0.5?l each ofprimers CDV-F8 and CDV-R8 (50pmol/?l) [40] was added to a totalof 49?l of the reaction mixture containing 0.2mM of each dNTP,1.2mM MgSO4and 1?l of a mix of SuperScript II H-Reverse Tran-scriptase and Platinum Taq HiFi (Invitrogen – Life Technologies,Milan, Italy). The RNA was reverse transcribed and immediatelysubjected to PCR amplification in a single-step protocol, usingSuperScript One-Step RT-PCR kit (Invitrogen – Life Technologies,Milan, Italy). Reverse transcription was carried out at 48◦C for60min, followed by denaturation of the reverse transcriptase at95◦C for 2min. Amplification was conducted by a temperaturecycling protocol consisting of 35 cycles of 30s of denaturation at94◦C, 1min of primer annealing at 55◦C, and 1min of extensionat 68◦C, followed by 10min of the final extension phase at 68◦C.To obtain PCR products suitable for sequencing, the inner primerpair RH-3 and RH-4 [40] was used to amplify in a nested PCR thecomplete H-gene. About 0.5?l each of primers RH-3 and RH-4(50pmol/?l) was added to a total of 49?l of the reaction mixtureconsisting of 0.5?l of a 1:100 dilution of the first-round PCR prod-uct, 0.25?l of TaKaRa LA Taq (5U/?l; Takara Bio Inc., Japan), and5?l of 10× PCR buffer, 8?l of 2.5mM dNTP mixture, and 35.75?lof distilled H2O. Amplification was conducted by a temperaturecycling protocol consisting of 25 cycles of 1min of denaturationat 94◦C, 2min of primer annealing at 50◦C, and 2min of exten-sion at 68◦C, followed by 2min of the final extension phase at68◦C.1.3. SequencingThe RH3–RH4 PCR products were purified with the Wizard PCRPreps DNA Purification System (Promega, Madison, WI). Then, theDNA was used as template for direct sequencing. The DNA wassequenced by using the conserved primers RH3 and RH4 and spe-cific primers designed according to an overlapping strategy. Thesequences were assembled using Bioedit software package ver-sion 2.1 [25] and compared to cognate sequences in the geneticdatabases using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) andFASTA (http://www.ebi.ac.uk/fasta33) web-based programs. Thegene H sequences of the strains Rocborn-46th and Rockborn-Candur is available under accession numbers GU810819 andGU266280.1.4. Phylogenetic analysisNucleic acid sequences were aligned with a selection of CDVH sequences by using Mega 4.0 software package [48]. The treewas inferred using the Kimura two-parameter model and theneighbor-joining method. Statistical significance of the phylogenywas estimated by bootstrap analysis over 1000 pseudoreplicatedata sets.2. Results and discussionThe H gene of the strain Rockborn-46th was 1824 nt long andcoded for a 607 aa-long polypeptide. Eight conserved potentialN-glycosylation sites were found at residues 19, 149, 309, 391,422, 456, 587 and 603. The virus Rockborn-46th differed in a

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1224V. Martella et al. / Vaccine 29 (2011) 1222–1227Fig. 1. Phylogenetic tree based on the full-length H gene displaying the genetic relationships between the strain Rockborn and a selection of CDV strains of various genotypes.Bootstrap values lower than 80% are not shown. In the box, a sub-tree generated using a 385-nt fragment of the H gene (nt 416–800 of the H gene) is shown.unique aa change, 544-K→T (nt 1631-A→C) from the vaccinevirus Rockborn-Candur. By FASTA analysis, the virus had the high-est identity (99.6% nt and 99.3% aa) to a CDV strain detected from aLesser Panda (Ailurus fulgens) in China (AF178039). In addition, thevirus was found to match (>99.0% nt identity in the full-length Hgene)acaninevirus(strain25259,accessionAY964114)detectedinMissouri2004[43]andtobevirtuallyidentical(100%nt)ina995-ntlongfragmenttoacaninestraindetectedinJapan1991(strain6083,accession AB191310) [50] and in a 800-nt long fragment to canineviruses (99.9–100% nt) detected in Austria 2007 (strain 2727-07and 2730-07, accessions GQ214370 and GQ214371, respectively).By phylogenetic analysis, using a selection of CDV strains repre-sentative of the various CDV lineages, Rockborn-like viruses wereshown to be genetically related to America-2 viruses and distantlyrelated to America-1 CDVs (Fig. 1). The strain Rockborn-46th pas-sage displayed 93.0% nt and 91.7% aa identity to the vaccine strainOnderstepoort, prototype of the America-1 lineage.Interestingly, the CDV strains from vaccines A and B were alsofound to contain Rockborn-like viruses. Vaccine A differed in onlytworesidues,160-R→Kand524-T→K,fromstrainRockborn-46thwhile vaccine B differed in 4 residues, 160-R→K, 303-L→S, 544-K→T and 589-T→P. The residue 544-T was also found in theRockborn-Candur strain, while the change 589-T→P in vaccineB disrupted the N-587-glycosylation site (Table 1). Assuming thatthe viruses likely came from different virus passages and batches,it is tempting to speculate that those differences may have beenaccumulated during cultivation on cell cultures. Accordingly, thelaboratory strain Rockborn 46th and the vaccine virus Rockborn-Candur appeared to be similar cell passages of the virus, sincethey differed only in a unique non-silent nt change. Similarly, vac-cine A differed in only two aa residues from the laboratory strainRockborn-46th; in contrast, vaccine B displayed 4 mutations, withthe change 589-T→P, altering a potential N-linked sugar bindingsite. By solving the crystal structure of measles virus H protein,

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V. Martella et al. / Vaccine 29 (2011) 1222–12271225Table 1Nucleotide and amino acid changes in the H gene and protein of CDV strains Rockborn-46th, Rockborn-Candur and Rockborn-like viruses. Amino acid mutations are indicated in brackets.Position nt4960174229297314479794796817903908966993102610561072111011121123117312301235157116311654–517581765Position aa7795160265266273303358370371375412524544552589Rockborn-46thAATG (D)TT (L)G (R)T (L)C (L)G (V)CT (L)ATAGA (I)A (K)A (Q)A (K)CAC (P)C (T)A (K)AG (R)CA (T)Rockborn-CandurC (T)Vaccine AA (K)A (K)Vaccine BA (K)C (S)C (T)C (P)Lesser PandaCA (N)CC (S)C (G)CA (Q)25259/US/04CGT (F)C (L)TCCCTG (V)C (N)T (L)G (E)TGA (H)TC (P)a morbillivirus closely related to CDV, sugar moieties have beenseen to mask massively the protein surface and to help the homod-imeric structure of the H protein to maintain correct orientation[29]. Residues of the putative receptor-binding site are strategi-cally positioned in an unshielded area on the top of the protein,whereneutralizingepitopeshavealsobeenmappedto[38,47],andtend to be highly conserved among morbilliviruses. Accordingly,changes in the sugar moieties can mask or expose epitopes on theH protein but functional constraints tend to conserve the antigenicmonotypic profile of morbilliviruses [29].An alternative hypothesis to explain the presence of Rockborn-like viruses in vaccines A and B is that they are variants of a fieldRockborn-like strain, circulating not only in Sweden in the 1950s,but also in other geographical settings and isolated on more occa-sions. By comparing the sequence of the virus Rockborn-46th withthe databases, at least five Rockborn-like sequences were identi-fied. Scattered mutations were identified in the full-length H genesequencesbetweenthevirusRockborn-46thandtheChineseLesserPanda virus (7 nt and 4 aa changes) and the USA strain 25259 (18nt and 8 aa changes) (Table 1). Due to the consistent number ofmutations, the origin of these viruses may not be unequivocallyattributed to Rockborn-based vaccines and a possible explanationis that Rockborn-like viruses are still circulating in the field orthat animalised vaccine-derived viruses were introduced in unre-lated geographical settings on several occasions and circulated inthe area in different carnivores, taking advantage of the broadhost range of CDV. Also, Ondestepoort-like CDVs, identified inthe 1930s and long believed to be extinct in the field, have beenidentified from outbreaks in wildlife animals in United States andhave been reported occasionally in dogs from Ireland, Korea andPoland [28,32,34,46], suggesting vaccine-induced disease, eventu-ally followed by animalisation of vaccine viruses in some animalpopulations, or persistence of America-1 CDVs in certain ecologicalsettings.Conversely, exploring the databases, three CDV sequences (oneJapanese and two Austrian viruses) were found to match (nearly100% nt) the sequence of strain Rockborn-46th. Such high geneticidentity is much more consistent with a hypothesis of residualvirulence of vaccine viruses. This phenomenon appears to be mod-ulated by host factors, such as age, genetic constitution, state ofimmunecompetenceand/orimmuno-suppressionduetoconcomi-tant diseases/infection as well as by virus-intrinsic factors, i.e.the grade of virus attenuation. Vaccine-related disease has beenreported on several occasions in dogs [4,14,18,23,27,33,39]. Pupsbecoming ill within 2–3 weeks after administration of CDV vaccineare always suspected to have developed vaccine-induced diseaseand this may result in disputes involving veterinary practition-ers, pet owners and commercial pet shops. In most suspectedcases, however, it has been difficult to establish whether the dis-ease was induced by the vaccine virus or by field viruses infectingpups shortly before or after vaccine administration. This issue wasnot ruled out firmly in older studies, since appropriate diagnos-tic tools were not available. Therefore, in some circumstances themagnitude of this phenomenon may have been overrated. Dur-ing a 2-year-surveillance in Italy, we found all suspected casesof CDV vaccine-induced illness or death to be caused by CDVfield strains [36], thus suggesting that residual virulence is infre-quent.The vaccine strain Rockborn was suspected to retain resid-ual virulence after several reports on vaccine-related disease[4,14,18,27] and this was attributed to an intrinsic virulence of lowpassage stocks established on primary dog kidney (DK) cells andgrown for vaccine passage cultures on Madin Darby Canine Kidney(MDCK) cells.In most cases, increased virulence of the dog-kidney-cell-attenuated CDV was suspected to have been triggered by the